Non-toxigenic Clostridium difficile spores for use in oral vaccination

ABSTRACT

Described are non-toxigenic  Clostridium difficile  strains and spores. Also described are vaccines comprising the  Clostridium difficile  spores. Further described are methods of preventing or treating a  Clostridium difficile  infection in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation application of U.S. patentapplication Ser. No. 15/905,598, filed on Feb. 26, 2018, which claimspriority to U.S. Provisional Patent Application No. 62/463,497, filed onFeb. 24, 2017, U.S. Provisional Patent Application No. 62/513,247, filedon May 31, 2017, and U.S. Provisional Patent Application No. 62/588,777,filed on Nov. 20, 2017, each of which are incorporated herein byreference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Numbers NIHR21 AI113470 and NIH K01 DK092352 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 29,012 Byte ASCII (Text) file named“17A102PRC-210112-9081-US02-SEQ-LIST-02-26-18.txt,” created on Feb. 26,2018.

PARTIES TO A JOINT RESEARCH AGREEMENT

Inventions disclosed or claimed herein were made pursuant to a JointResearch Agreement between the University of South Florida and Trusteesof Tufts College.

TECHNICAL FIELD

The present disclosure relates to genetically modified non-toxigenicClostridium difficile strains, spores, vaccinations, and methods fortreating and/or preventing infections caused by Clostridium difficile.

BACKGROUND

Clostridium difficile is a spore-forming anaerobic and toxin-producingbacillus. It is the most common cause of nosocomialantibiotic-associated diarrhea. A CDC study estimated that 29,000 deathswere caused by Clostridium difficile in the U.S. in 2011. Antibiotictreatment of Clostridium difficile infections may be difficult, due bothto antibiotic resistance and physiological factors of the bacteria(e.g., spore formation and protective effects of the pseudomembrane).Accordingly, there exists a need for effective therapies and preventionof infections caused by Clostridium difficile.

SUMMARY OF THE INVENTION

In one aspect, disclosed is a non-toxigenic Clostridium difficile straincomprising an immunogenic protein, which comprises a glucosyltranferasedomain of Clostridium difficile toxin TcdB, a cysteine proteinase domainof Clostridium difficile toxin TcdB, and a receptor binding domain ofClostridium difficile toxin TcdA. The glucosyltranferase domain ofClostridium difficile toxin TcdB comprises a W102A amino acidsubstitution and a D288N amino acid substitution. Also disclosed in anon-toxigenic Clostridium difficile strain comprising aglucosyltranferase domain of Clostridium difficile toxin TcdB, acysteine proteinase domain of Clostridium difficile toxin TcdB, areceptor binding domain of Clostridium difficile toxin TcdA, and areceptor binding domain of Clostridium difficile toxin TcdB. Theglucosyltranferase domain of Clostridium difficile toxin TcdB comprisesa W102A amino acid substitution and a D288N amino acid substitution. TheClostridium difficile strains may form spores.

Also disclosed is a vaccine comprising the spores formed by theClostridium difficile strains. Further disclosed is a method of treatingor preventing Clostridium difficile bacterial infection in a subject inneed thereof. In some aspects, the vaccine is administered orally. Thevaccine increases levels of anti-TcdA and anti-TcdB IgG antibodies inthe subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the domains of TcdA and TcdB and construction of mTcd138and Tcd169. (A) Both toxins share similar domains, including theglucosyltransferase domain (GT), the cysteine proteinase domain (CPD),the translocation domain (TMD) and the receptor binding domain (RBD).The DXD motif and a conserved tryptophan in the GT are involved in theenzymatic activity. (B) mTcd138 (SEQ ID NO.:4) was constructed by fusingthe GT and CPD of TcdB (SEQ ID NO.:5) with the RBD of TcdA (SEQ ID NO:6)through the linker (SEQ ID NO.:1). Two point mutations were made in theGT of TcdB, which essentially eliminates the toxicity. (C) Tcd169 (SEQID NO.:3) was constructed by fusing the GT and CPD of TcdB (SEQ IDNO.:5), the RBD of TcdB (SEQ ID NO.:7) with the RBD of TcdA (SEQ IDNO:6) through the linker (SEQ ID NO.:1). Two point mutations were madein the GT of TcdB and one point mutation was made in the CPD of TcdB.

FIG. 2 shows the domains of Tcd138, which comprise theglucosyltransferase domain (GT) of TcdB, the cysteine proteinase domain(CPD) of TcdB, and the receptor binding domain (RBD) of TcdA.

FIG. 3 shows the domains of Tcd169, which comprise theglucosyltransferase domain (GT) of TcdB, the cysteine proteinase domain(CPD) of TcdB, the receptor binding domain (RBD) of TcdB, and thereceptor binding domain (RBD) of TcdA.

FIG. 4 shows the expression and purification of Tcd169. Gene sequenceencoding Tcd169 was synthesized and cloned in Bacillus megatarium.Protein Tcd169 was purified from bacterial lysate by Ni-affinitychromatography and gel filtration, and analyzed by SDS-PAGE.

FIG. 5 shows the protective responses of Tcd169 immunization (IM or IP)in mice. Groups of C57 BL/6 mice (n=10) were immunized with Tcd169 (10μg) or PBS in the presence of alum for 3 times at 14-day intervals (IMor IP). Anti-toxin IgG titers (A) and anti-toxin neutralizing titers (B,C) in sera from third immunization were measured. Seven days after thirdimmunization, mice were given antibiotic mixture in drinking water for 4days, switched to regular water for 2 days, and were given one dose ofclindamycin (10 mg/kg) one day before infection with 10⁶ of C. difficileUK6 spores by gavage. After infection, mouse survivals (P=0.0486 betweenPBS and Tcd169 IM/Tcd169 IP groups) (D), and mean relative weightchanges (E) of different groups were recorded. The neutralizing titer isexpressed as the maximum dilution of the sera that inhibits Vero cellrounding caused by toxin at a given concentration. This givenconcentration is the minimum toxin dose causing cell rounding after a 16h of toxin exposure, i.e., 2.5 and 0.1 ng/ml for TcdA and TcdB,respectively.

FIG. 6 shows the expression of mTcd138 in non-toxigenic C. difficile 85strain (NTCD). Western blot analysis of supernatants and pellets ofNTCD_Tcd138 and NTCD (mTcd138 as positive control) using anti-TcdA (A)or anti-TcdB (B) antibodies.

FIG. 7 shows that oral immunization of mice with NTCD_Tcd138 sporesinduces mucosal and systemic toxin-specific antibody responses. Groupsof C57 BL/6 mice (N=10) were orally immunized with NTCD_mTcd138 (2×10⁶spores/immunization for 3 times at 14-day intervals). Sera and feceswere collected after each immunization. Before use, feces were dissolved(0.1 g/ml) in PBS containing proteinase inhibitors. Anti-TcdA/anti-TcdBIgG titers in sera (A), anti-TcdA/anti-TcdB IgA titers in sera (B) or infeces (C) were determined by ELISA.

FIG. 8 shows anti-toxin neutralizing titers of sera or feces from miceorally immunized with NTCD_Tcd138 spores. Vero cells were used todetermine in vitro neutralizing activities of sera (A) or feces (B). Theneutralizing titer is expressed as the maximum dilution of the sera thatinhibits cell rounding caused by toxin at a given concentration. Thisgiven concentration is the minimum toxin dose causing cell roundingafter a 16 h of toxin exposure, i.e., 2.5 and 0.1 ng/ml for TcdA andTcdB, respectively.

FIG. 9 shows that oral immunization of mice with NTCD_Tcd138 or NTCDspores induces mucosal and systemic antibody responses against FliCD.Groups of C57 BL/6 mice (N=10) were orally immunized with NTCD_mTcd138or NTCD at 2×10⁶ spores for 3 times at 14-day intervals. Sera and feceswere collected after each immunization. Before use, feces were dissolved(0.1 g/ml) in PBS with proteinase inhibitors. Anti-FliCD IgG titers insera (A) or feces (C), and anti-FliCD IgA titers in sera (B) or feces(D) were determined by ELISA.

FIG. 10 shows that oral immunization with NTCD_Tcd138 spores providesfull protection to mice against infection with a hyper-virulent C.difficile strain UK6. Groups of mice (N=10) were orally immunized withNTCD, or NTCD_Tcd138 (2×10⁶ spores in 200 μl PBS) or PBS (200 μl) for 3times at 14-day intervals. Seven days after third immunization, micewere given antibiotic mixture in drinking water for 4-days, switched toregular water for 2 days, and were given one dose of clindamycin (10mg/kg) before infection with 10⁶ spores C. difficile UK6 by gavage. Micewere monitored, and mouse survivals (P=0.495 between groups PBS andNTCD; P=0.0002 between groups PBS and NTCD_Tcd138) (A), mean relativeweight changes (B) and frequency of diarrhea (C) of different groupswere recorded.

FIG. 11 shows fecal toxin levels of mice orally immunized with NTCD,NTCD_Tcd138 spores or PBS followed by infection with C. difficile UK6spores. Groups of mice (N=10) were orally immunized with NTCD, orNTCD_Tcd138 (2×10⁶ spores in 200 μl PBS) or PBS (200 μl) for 3 times at14-day intervals. Seven days after third immunization, mice were givenantibiotic mixture in drinking water for 4-days, switched to water fordays, and were given one dose of clindamycin (10 mg/kg) before infectionwith 10⁶ spores C. difficile UK6 by gavage. Feces were collected on postinfection days 0, 1, and 2, dissolved (0.1 g/ml) in PBS with proteinaseinhibitors. TcdA (A) or TcdB (B) levels in feces were determined byELISA.

FIG. 12 shows that oral immunization of hamsters with NTCD_Tcd138 sporesinduces systemic toxin-specific antibody responses. Groups of goldenSyrian hamsters (N=10) were orally immunized with NTCD_mTcd138 at 2×10⁶spores for 3 times at 14-day intervals. Sera and feces were collectedafter each immunization. Anti-TcdA/anti-TcdB IgG titers in sera weredetermined by ELISA.

FIG. 13 shows anti-toxin neutralizing titers of sera or feces fromhamsters orally immunized with NTCD_Tcd138 spores. Vero cells were usedto determine in vitro neutralizing activities of sera (A) or feces (B).The neutralizing titer is expressed as the maximum dilution of the serathat inhibits cell rounding caused by toxin at a given concentration.This given concentration is the minimum toxin dose causing cell roundingafter a 16 h of toxin exposure, i.e., 2.5 and 0.1 ng/ml for TcdA andTcdB, respectively.

FIG. 14 shows that oral immunization with NTCD_Tcd138 spores providessignificant protection to hamsters against infection with a virulent C.difficile UK6 strain at a dose of 200-fold of the lethal infection dose.Groups of hamsters (N=10) were orally immunized with NTCD, orNTCD_Tcd138 (2×10⁶ spores in 200 μl PBS) or PBS (200 μl) for 3 times at14-day intervals. Seven days after third immunization, hamsters weregiven clindamycin (IP, 40 mg/kg/day for 2 days), followed by infectionwith 2×10⁴ C. difficile UK1 spores by gavage. Hamsters were monitored,and survivals (P=0.0754 between groups PBS and NTCD; P=0.0453 betweengroups PBS and NTCD_Tcd138) (A) and diarrhea frequency (B) recorded.

FIG. 15 shows the toxin gene profiles of two selected C. difficilestrains. Lane 1, tcdA⁺, tcdB⁺; Lane 2, non-toxigenic C. difficile; LaneM: 100-bp DNA marker. A rapid 3-plex PCR was developed for the detectionof tcdA, tcdB and 16s rDNA. 5 μl of 12-24 hrs of C. difficile culturewas used as template.

FIG. 16 shows the colonic inflammation and injury caused by directintra-rectal instillation of TcdA/TcdB. A 5F infant feeding tube wasinserted 2.5 cm up the colon. 100 μl of TcdA (10 μg)+TcdB (10 μg) or PBSwas slowly administered. 4 or 5 hours later mice were euthanized anddissected to analyze the toxin-mediated effects on the colon.

FIG. 17 shows immunization and challenge schemes for CDI relapse modelsin mice. (A) After 3 immunizations mice will be pretreated withantibiotic mixture, challenged with C. difficile UK6 spores, andmonitored for about a week. Thirty days after initial spore challenge,survived mice will be again treated with antibiotics mixture followed byinfection with C. difficile UK6 spores and monitoring. (B) Non-immunizednaïve mice will be pretreated with antibiotic mixture, challenged withC. difficile UK6 spores, and monitored for about a week. Starting onpost-infection day 5, mice will be immunized for 3 times at 10-dayintervals. Ten days after third immunization, mice will be again treatedwith antibiotics mixture followed by infection with C. difficile UK6spores and monitoring.

FIG. 18 shows the nucleotide sequence that encodes for Tcd169 (4251 bp)(SEQ ID NO.: 2).

FIG. 19 shows the amino acid sequence for Tcd169 (SEQ ID NO.: 3).

FIG. 20 shows the amino acid sequence for Tcd138 (SEQ ID NO.: 4).

DETAILED DESCRIPTION

Disclosed herein are genetically modified non-toxigenic Clostridiumdifficile strains, spores, vaccinations, and methods for treating and/orpreventing bacterial infections. The bacterial infections may be causedby Clostridium difficile. The disclosed Clostridium difficile spores maybe used to prevent a Clostridium difficile infection in a subject. Thedisclosed Clostridium difficile strains may be used to treat aClostridium difficile infection in a subject.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (forexample, it includes at least the degree of error associated with themeasurement of the particular quantity). The modifier “about” shouldalso be considered as disclosing the range defined by the absolutevalues of the two endpoints. For example, the expression “from about 2to about 4” also discloses the range “from 2 to 4.” The term “about” mayrefer to plus or minus 10% of the indicated number. For example, “about10%” may indicate a range of 9% to 11%, and “about 1” may mean from0.9-1.1. Other meanings of “about” may be apparent from the context,such as rounding off, so, for example “about 1” may also mean from 0.5to 1.4.

The terms “administration” or “administering” as used herein may includethe process in which the compositions and vaccines as described herein,alone or in combination with other compounds or compositions, aredelivered to a subject. The Clostridium difficile spores may beadministered in various routes including, but not limited to, oral,mucosal, mucosal nasal, parenteral (including intravenous,intra-arterial, and other appropriate parenteral routes), intrathecally,intramuscularly, subcutaneously, colonically, rectally, and nasally,transcutaneously, among others. The dosing of the Clostridium difficilespores described herein to obtain a therapeutic or prophylactic effectmay be determined by the circumstances of the subject, as known in theart. The dosing of a subject herein may be accomplished throughindividual or unit doses of the Clostridium difficile spores herein orby a combined or prepackaged or pre-formulated dose of the Clostridiumdifficile strains.

Administration may depend upon the amount of Clostridium difficilespores administered, the number of doses, and duration of treatment. Forexample, multiple doses of the Clostridium difficile spores may beadministered. The frequency of administration of the compositions andvaccines may vary depending on any of a variety of factors. The durationof administration of the Clostridium difficile spores, e.g., the periodof time over which the Clostridium difficile spores are administered,may vary, depending on any of a variety of factors, including subjectresponse, etc.

The amount of the Clostridium difficile spores administered may varyaccording to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, idiosyncraticresponses of the individual, the dosimetry, and the like. Detectablyeffective amounts of the Clostridium difficile spores of the presentdisclosure may also vary.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used in this specification and theappended claims, the term “or” is generally employed in its senseincluding “and/or” unless the context clearly dictates otherwise.

The term “bacterial strain” and “strain” as used herein, refer to agenetic variant, genetically modified, or subtype of bacteria. Thestrain may be a genetically modified form of Clostridium difficile.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “an” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

The terms “Clostridium difficile”, “C. difficile”, “C. diff”, and “CDF”,and “cdf” as used herein, may be used interchangeably.

The term “genetically modified” as used herein, refers to geneticmaterial that has been altered using genetic engineering techniques. Anorganism may be genetically modified. Bacteria may be geneticallymodified.

The term “immunogen”, as used herein refers to any substance that may bespecifically bound by components of the immune system.

The term “nanoparticle” as used herein refers to particles that arebetween 1 and 100 nanometers in size.

The term “parenterally,” as used herein, refers to modes ofadministration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” or “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and/oradjuvant that are useful in preparing a pharmaceutical composition thatare generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use and/or human pharmaceutical use.“A pharmaceutically acceptable excipient, diluent, carrier and/oradjuvant” as used herein includes one or more such excipients, diluents,carriers, and adjuvants.

As used herein, the term “spore” includes, but is not limited to, astructure produced by bacteria that is resistant to many environmentalor induced factors. The spore may be a Clostridium difficile spore.

As used herein, the term “subject,” “patient,” or “organism” includeshumans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses).Typical subjects to which an agent(s) of the present disclosure may beadministered may include mammals, particularly primates, especiallyhumans. For veterinary applications, suitable subjects may include, forexample, livestock such as cattle, sheep, goats, cows, swine, and thelike; poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, suitable subjects may includemammals, such as rodents (e.g., mice, rats, hamsters), rabbits,primates, and swine such as inbred pigs and the like. The subject mayhave a bacterial infection. The subject may have a bacterial infectioncaused by Clostridium difficile. The subject may be taking antibiotics.The subject may be taking antibiotics for a bacterial infection that iscaused by bacteria other than Clostridium difficile. The subject may beat risk for an infection caused by Clostridium difficile.

The “therapeutically effective amount” for purposes herein may bedetermined by such considerations as are known in the art. Atherapeutically effective amount of a compound may include the amountnecessary to provide a therapeutically effective result in vivo. Theamount of the compound or composition must be effective to achieve aresponse, including but not limited to a total prevention of (e.g.,protection against) of a condition, improved survival rate or more rapidrecovery, improvement or elimination of symptoms associated with thecondition (such as cancer), or other indicators as are selected asappropriate measures by those skilled in the art. As used herein, asuitable single dose size includes a dose that is capable of preventingor alleviating (reducing or eliminating) a symptom in a subject whenadministered one or more times over a suitable time period. The“therapeutically effective amount” of a compound or composition asdescribed herein may depend on the route of administration, type ofsubject being treated, and the physical characteristics of the subject.These factors and their relationship to dose are well known to one ofskill in the medicinal art, unless otherwise indicated.

The term “toxin” as used herein, may refer to small molecules, peptides,or proteins that are capable of causing disease on contact with orabsorption by body tissues interacting with biological macromoleculessuch as enzymes or cellular receptors. Toxins may be produced bymicroorganisms. Toxins may be produced by Clostridium difficile. Toxinsmay be virulence determinants responsible for microbial pathogenicity.Toxins may be virulence determinants responsible for evasion of the hostimmune response.

As used herein, “treat”, “treatment”, “treating”, and the like refer toacting upon a condition with an agent to affect the condition byimproving or altering it. The condition includes, but is not limited toinfection, such as those caused by bacteria. The bacterial infection maybe caused by Clostridium difficile. The aforementioned terms cover oneor more treatments of a condition in a subject (e.g., a mammal,typically a human or non-human animal of veterinary interest), andinclude: (a) reducing the risk of occurrence of the condition in asubject determined to be predisposed to the condition but not yetdiagnosed, (b) impeding the development of the condition, and/or (c)relieving the condition, e.g., causing regression of the conditionand/or relieving one or more condition symptoms (e.g., treatingbacterial infections caused by Clostridium difficile).

As used herein, the term “virulence factors” include, but are notlimited to the ability of bacteria to cause disease in terms of thenumber of infecting bacteria, the route of entry into the body, theeffects of host defense mechanisms, and intrinsic characteristics of thebacteria called. A “hyper-virulent” bacterial strain may be moredifficult to treat. A “hyper-virulent” bacterial strain may causesymptoms that are worse than an infection from a bacterial strain thatis not hyper-virulent. A “hyper-virulent” bacterial strain may be moredeadly.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. IMMUNOGENIC PROTEINS

Pathogenic Clostridium difficile infection strains produce multipletoxins. Clostridium difficile produces toxins. Two Clostridium difficileinfection toxins are enterotoxin (Clostridium difficile toxin A (TcdA))and cytotoxin (Clostridium difficile toxin B (TcdB)). Toxins A and B areglucosyltransferases that target and inactivate the Rho family ofGTPases. TcdB may induce actin depolymerization by a mechanismcorrelated with a decrease in the ADP-ribosylation of the low molecularmass GTP-binding Rho proteins.

In one aspect, disclosed are immunogenic proteins that comprise one ormore domains from Clostridium difficile toxins. The immunogenic proteinmay be a chimeric protein. The immunogenic protein may comprise one ormore domains from Clostridium difficile toxin A (TcdA). The immunogenicprotein may comprise the glucosyltransferase domain (GT) from TcdA. Theimmunogenic protein may comprise the cysteine proteinase domain (CPD)from TcdA. The immunogenic protein may comprise the receptor bindingdomain (RBD) from TcdA. The immunogenic protein may contain one or moredomains from Clostridium difficile toxin B (TcdB). The immunogenicprotein may comprise the glucosyltransferase domain (GT) from TcdB. Theimmunogenic protein may comprise the cysteine proteinase domain (CPD)from TcdB. The immunogenic protein may comprise the receptor bindingdomain (RBD) from TcdB.

The one or more domains may be connected by an amino acid linker. Theamino acid linker may be the amino acid sequence of GGSG (SEQ ID NO.:1).

In some embodiments, the immunogenic protein comprises theglucosyltransferase domain of TcdB, cysteine proteinase domain of TcdB,receptor binding domain of TcdB, and the receptor binding domain ofTcdA. The domains may be connected by an amino acid linker. The linkermay be the amino acid linker GGSG. In some embodiments, there is a W102Aamino acid substitution and a D288N amino acid substitution in the GT ofTcdB and a C698A amino acid substitution in the CPD of TcdB. The W102Aamino acid substitution and D288N amino acid substitution in the GT ofTcdB eliminate toxicity of TcdB. The immunogenic protein may be encodedby the nucleotide sequence as set forth in SEQ ID NO.: 2. Theimmunogenic protein may be Tcd169 (SEQ ID NO.: 3).

In some embodiments, the immunogenic protein comprises theglucosyltransferase domain of TcdB, the cysteine proteinase domain ofTcdB, and the receptor binding domain (RBD) of TcdA. The domains may beconnected by an amino acid linker. The linker may be the amino acidlinker GGSG. In some embodiments, there is a W102A amino acidsubstitution and a D288N amino acid substitution in the GT of TcdB and aC698A amino acid substitution in the CPD of TcdB. The W102A amino acidsubstitution and D288N amino acid substitution in the GT of TcdBeliminate toxicity of TcdB. The immunogenic protein may be Tcd138 (SEQID NO.: 4). The immunogenic protein may be encoded by a nucleotidesequence that encodes Tcd138 (SEQ ID NO.: 4).

3. NON-TOXIGENIC CLOSTRIDIUM DIFFICILE STRAINS

In one aspect, disclosed are non-toxigenic Clostridium difficilestrains. The non-toxigenic Clostridium difficile strain may beClostridium difficile 85 strain (NTCD). In one aspect, disclosed aregenetically modified non-toxigenic Clostridium difficile strains. Thenon-toxigenic Clostridium difficile strain may comprise immunogenicproteins that comprise one or more domains from Clostridium difficiletoxins. The non-toxigenic Clostridium difficile strain may comprise theimmunogenic protein Tcd169. The non-toxigenic Clostridium difficilestrain may be NTCD_Tcd169. The non-toxigenic Clostridium difficilestrain may comprise the immunogenic protein Tcd138. The non-toxigenicClostridium difficile strain may be NTCD_Tcd138. The disclosednon-toxigenic Clostridium difficile strains may form spores. The sporesmay be administered to a subject to treat or prevent a Clostridiumdifficile infection.

4. PHARMACEUTICAL COMPOSITIONS

The disclosed Clostridium difficile spores may be incorporated intopharmaceutical compositions suitable for administration to a subject(such as a patient, which may be a human or non-human).

The pharmaceutical compositions may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of the agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thecomposition may be determined by a person skilled in the art and mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the composition to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of Clostridiumdifficile spores of the disclosure (e.g., a composition and vaccine) areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

For example, a therapeutically effective amount of disclosed Clostridiumdifficile spores may be, but is not limited to, about 1 mg/kg to about1000 mg/kg, about 5 mg/kg to about 950 mg/kg, about 10 mg/kg to about900 mg/kg, about 15 mg/kg to about 850 mg/kg, about 20 mg/kg to about800 mg/kg, about 25 mg/kg to about 750 mg/kg, about 30 mg/kg to about700 mg/kg, about 35 mg/kg to about 650 mg/kg, about 40 mg/kg to about600 mg/kg, about 45 mg/kg to about 550 mg/kg, about 50 mg/kg to about500 mg/kg, about 55 mg/kg to about 450 mg/kg, about 60 mg/kg to about400 mg/kg, about 65 mg/kg to about 350 mg/kg, about 70 mg/kg to about300 mg/kg, about 75 mg/kg to about 250 mg/kg, about 80 mg/kg to about200 mg/kg, about 85 mg/kg to about 150 mg/kg, and about 90 mg/kg toabout 100 mg/kg.

The pharmaceutical compositions may include pharmaceutically acceptablecarriers. The term “pharmaceutically acceptable carrier,” as usedherein, means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as, but not limited to, lactose,glucose and sucrose; starches such as, but not limited to, corn starchand potato starch; cellulose and its derivatives such as, but notlimited to, sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as, but not limited to, cocoa butter and suppository waxes; oilssuch as, but not limited to, peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil and soybean oil; glycols; such aspropylene glycol; esters such as, but not limited to, ethyl oleate andethyl laurate; agar; buffering agents such as, but not limited to,magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol, and phosphatebuffer solutions, as well as other non-toxic compatible lubricants suchas, but not limited to, sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by, for example, soliddosing, eyedrop, in a topical oil-based formulation, injection,inhalation (either through the mouth or the nose), implants, or oral,buccal, parenteral, or rectal administration. Techniques andformulations may generally be found in “Remington's PharmaceuticalSciences”, (Meade Publishing Co., Easton, Pa.). Therapeutic compositionsmust typically be sterile and stable under the conditions of manufactureand storage.

The route by which the disclosed Clostridium difficile spores areadministered and the form of the composition will dictate the type ofcarrier to be used. The composition may be in a variety of forms,suitable, for example, for systemic administration (e.g., oral, rectal,nasal, sublingual, buccal, implants, or parenteral) or topicaladministration (e.g., dermal, pulmonary, nasal, aural, ocular, liposomedelivery systems, transdermal, or iontophoresis).

Carriers for systemic administration typically include at least one ofdiluents, lubricants, binders, disintegrants, colorants, flavors,sweeteners, antioxidants, preservatives, glidants, solvents, suspendingagents, wetting agents, surfactants, combinations thereof, and others.All carriers are optional in the compositions.

Suitable diluents include sugars such as glucose, lactose, dextrose, andsucrose; diols such as propylene glycol; calcium carbonate; sodiumcarbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. Theamount of diluent(s) in a systemic or topical composition is typicallyabout 50 to about 90%.

Suitable lubricants include silica, talc, stearic acid and its magnesiumsalts and calcium salts, calcium sulfate; and liquid lubricants such aspolyethylene glycol and vegetable oils such as peanut oil, cottonseedoil, sesame oil, olive oil, corn oil and oil of theobroma. The amount oflubricant(s) in a systemic or topical composition is typically about 5to about 10%.

Suitable binders include polyvinyl pyrrolidone; magnesium aluminumsilicate; starches such as corn starch and potato starch; gelatin;tragacanth; and cellulose and its derivatives, such as sodiumcarboxymethylcellulose, ethyl cellulose, methylcellulose,microcrystalline cellulose, and sodium carboxymethylcellulose. Theamount of binder(s) in a systemic composition is typically about 5 toabout 50%.

Suitable disintegrants include agar, alginic acid and the sodium saltthereof, effervescent mixtures, croscarmelose, crospovidone, sodiumcarboxymethyl starch, sodium starch glycolate, clays, and ion exchangeresins. The amount of disintegrant(s) in a systemic or topicalcomposition is typically about 0.1 to about 10%.

Suitable colorants include a colorant such as an FD&C dye. When used,the amount of colorant in a systemic or topical composition is typicallyabout 0.005 to about 0.1%.

Suitable flavors include menthol, peppermint, and fruit flavors. Theamount of flavor(s), when used, in a systemic or topical composition istypically about 0.1 to about 1.0%.

Suitable sweeteners include aspartame and saccharin. The amount ofsweetener(s) in a systemic or topical composition is typically about0.001 to about 1%.

Suitable antioxidants include butylated hydroxyanisole (“BHA”),butylated hydroxytoluene (“BHT”), and vitamin E. The amount ofantioxidant(s) in a systemic or topical composition is typically about0.1 to about 5%.

Suitable preservatives include benzalkonium chloride, methyl paraben andsodium benzoate. The amount of preservative(s) in a systemic or topicalcomposition is typically about 0.01 to about 5%.

Suitable glidants include silicon dioxide. The amount of glidant(s) in asystemic or topical composition is typically about 1 to about 5%.

Suitable solvents include water, isotonic saline, ethyl oleate,glycerine, hydroxylated castor oils, alcohols such as ethanol, andphosphate buffer solutions. The amount of solvent(s) in a systemic ortopical composition is typically from about 0 to about 100%.

Suitable suspending agents include AVICEL RC-591 (from FMC Corporationof Philadelphia, Pa.) and sodium alginate. The amount of suspendingagent(s) in a systemic or topical composition is typically about 1 toabout 8%.

Suitable surfactants include lecithin, Polysorbate 80, and sodium laurylsulfate, and the TWEENS from Atlas Powder Company of Wilmington, Del.Suitable surfactants include those disclosed in the C.T.F.A. CosmeticIngredient Handbook, 1992, pp. 587-592; Remington's PharmaceuticalSciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1,Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. Theamount of surfactant(s) in the systemic or topical composition istypically about 0.1% to about 5%.

Although the amounts of components in the systemic compositions may varydepending on the type of systemic composition prepared, in general,systemic compositions include 0.01% to 50% of active and 50% to 99.99%of one or more carriers. Compositions for parenteral administrationtypically include 0.1% to 10% of actives and 90% to 99.9% of a carrierincluding a diluent and a solvent.

Compositions for oral administration can have various dosage forms. Theoral dosage form may be a vaccination. The oral dosage form may be solidforms including tablets, capsules, granules, and bulk powders. Theseoral dosage forms include a safe and effective amount, usually at leastabout 5%, and more particularly from about 25% to about 50% of actives.The oral dosage compositions include about 50% to about 95% of carriers,and more particularly, from about 50% to about 75%.

Tablets can be compressed, tablet triturates, enteric-coated,sugar-coated, film-coated, or multiple-compressed. Tablets typicallyinclude an active component, and a carrier comprising ingredientsselected from diluents, lubricants, binders, disintegrants, colorants,flavors, sweeteners, glidants, and combinations thereof. Specificdiluents include calcium carbonate, sodium carbonate, mannitol, lactoseand cellulose. Specific binders include starch, gelatin, and sucrose.Specific disintegrants include alginic acid and croscarmelose. Specificlubricants include magnesium stearate, stearic acid, and talc. Specificcolorants are the FD&C dyes, which can be added for appearance. Chewabletablets preferably contain sweeteners such as aspartame and saccharin,or flavors such as menthol, peppermint, fruit flavors, or a combinationthereof.

Capsules (including implants, time release and sustained releaseformulations) typically include an active compound, and a carrierincluding one or more diluents disclosed above in a capsule comprisinggelatin. Granules typically comprise a disclosed compound, andpreferably glidants such as silicon dioxide to improve flowcharacteristics. Implants can be of the biodegradable or thenon-biodegradable type.

The selection of ingredients in the carrier for oral compositionsdepends on secondary considerations like taste, cost, and shelfstability, which are not critical for the purposes of this invention.

Solid compositions may be coated by conventional methods, typically withpH or time-dependent coatings, such that a disclosed compound isreleased in the gastrointestinal tract in the vicinity of the desiredapplication, or at various points and times to extend the desiredaction. The coatings typically include one or more components selectedfrom the group consisting of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethylcellulose, EUDRAGIT coatings (available from Rohm & Haas G.M.B.H. ofDarmstadt, Germany), waxes and shellac.

Compositions for oral administration can have liquid forms. For example,suitable liquid forms include aqueous solutions, emulsions, suspensions,solutions reconstituted from non-effervescent granules, suspensionsreconstituted from non-effervescent granules, effervescent preparationsreconstituted from effervescent granules, elixirs, tinctures, syrups,and the like. Liquid compositions, which may be administered orally, mayinclude a disclosed compositions, and vaccines and a carrier, namely, acarrier selected from diluents, colorants, flavors, sweeteners,preservatives, solvents, suspending agents, and surfactants. Peroralliquid compositions preferably include one or more ingredients selectedfrom colorants, flavors, and sweeteners.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically include one or more of soluble filler substancessuch as diluents including sucrose, sorbitol and mannitol; and binderssuch as acacia, microcrystalline cellulose, carboxymethyl cellulose, andhydroxypropyl methylcellulose. Such compositions may further includelubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

The disclosed Clostridium difficile spores may be topicallyadministered. Topical compositions that can be applied locally to theskin may be in any form including solids, solutions, oils, creams,ointments, gels, lotions, shampoos, leave-on and rinse-out hairconditioners, milks, cleansers, moisturizers, sprays, skin patches, andthe like. The carrier of the topical composition preferably aidspenetration of the compounds into the skin. The carrier may furtherinclude one or more optional components. Transdermal administration maybe used to facilitate delivery.

The amount of the carrier employed in conjunction with a disclosedcompound is sufficient to provide a practical quantity of compositionfor administration per unit dose of the medicament. Techniques andcompositions for making dosage forms useful in the methods of thisinvention are described in the following references: ModernPharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979);Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); andAnsel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

A carrier may include a single ingredient or a combination of two ormore ingredients. In the topical compositions, the carrier includes atopical carrier. Suitable topical carriers include one or moreingredients selected from phosphate buffered saline, isotonic water,deionized water, monofunctional alcohols, symmetrical alcohols, aloevera gel, allantoin, glycerin, vitamin A and E oils, mineral oil,propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castoroil, combinations thereof, and the like. More particularly, carriers forskin applications include propylene glycol, dimethyl isosorbide, andwater, and even more particularly, phosphate buffered saline, isotonicwater, deionized water, monofunctional alcohols, and symmetricalalcohols.

The carrier of a topical composition may further include one or moreingredients selected from emollients, propellants, solvents, humectants,thickeners, powders, fragrances, pigments, and preservatives, all ofwhich are optional.

Suitable emollients include stearyl alcohol, glyceryl monoricinoleate,glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil,cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate,isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate,decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate,di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropylstearate, butyl stearate, polyethylene glycol, triethylene glycol,lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylatedlanolin alcohols, petroleum, mineral oil, butyl myristate, isostearicacid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyllactate, decyl oleate, myristyl myristate, and combinations thereof.Specific emollients for skin include stearyl alcohol andpolydimethylsiloxane. The amount of emollient(s) in a skin-based topicalcomposition is typically about 5% to about 95%.

Suitable propellants include propane, butane, isobutane, dimethyl ether,carbon dioxide, nitrous oxide, and combinations thereof. The amount ofpropellant(s) in a topical composition is typically about 0% to about95%.

Suitable solvents include water, ethyl alcohol, methylene chloride,isopropanol, castor oil, ethylene glycol monoethyl ether, diethyleneglycol monobutyl ether, diethylene glycol monoethyl ether,dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinationsthereof. Specific solvents include ethyl alcohol and homotopic alcohols.The amount of solvent(s) in a topical composition is typically about 0%to about 95%.

Suitable humectants include glycerin, sorbitol, sodium2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate,gelatin, and combinations thereof. Specific humectants include glycerin.The amount of humectant(s) in a topical composition is typically 0% to95%.

The amount of thickener(s) in a topical composition is typically about0% to about 95%.

Suitable powders include beta-cyclodextrins, hydroxypropylcyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums,colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammoniumsmectites, trialkyl aryl ammonium smectites, chemically-modifiedmagnesium aluminum silicate, organically-modified Montmorillonite clay,hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodiumcarboxymethyl cellulose, ethylene glycol monostearate, and combinationsthereof. The amount of powder(s) in a topical composition is typically0% to 95%.

The amount of fragrance in a topical composition is typically about 0%to about 0.5%, particularly, about 0.001% to about 0.1%.

Suitable pH adjusting additives include HCl or NaOH in amountssufficient to adjust the pH of a topical pharmaceutical composition.

In an embodiment, the pharmaceutical composition may include humanbreast milk. The active pharmaceutical ingredient may be a component ofhuman breast milk. The human breast milk may thus be administered to asubject in need of the active pharmaceutical ingredient.

5. METHOD OF TREATMENT AND METHOD OF PREVENTING BACTERIAL INFECTION

The disclosed Clostridium difficile spores may be used in methods fortreatment of bacterial infections. The Clostridium difficile spores asdisclosed herein may be used for preventing bacterial infections in asubject. The methods of treatment may comprise administering to asubject in need of such treatment, a composition comprising atherapeutically effective amount of the Clostridium difficile sporesdisclosed herein. Spores formed by the NTCD_Tcd169 strain may beadministered to treat or prevent Clostridium difficile infection. Sporesformed by the NTCD_Tcd138 strain may be administered to treat or preventClostridium difficile infection. Spores formed by the NTCD_Tcd169 strainmay be administered to treat or prevent Clostridium difficile infection.Spores formed by the NTCD_Tcd169 strain may be administered incombination with spores formed by the NTCD_Tcd138 strain to treat orprevent Clostridium difficile infection. The Clostridium difficileinfection treated by the methods disclosed herein may be caused by ahyper-virulent Clostridium difficile strain

Clostridium difficile colonization may be targeted by the administrationof the Clostridium difficile spores disclosed herein. Clostridiumdifficile growth factors may be targeted by the administration of theClostridium difficile spores disclosed herein. Clostridium difficiletoxins may be targeted by the administration of the Clostridiumdifficile spores disclosed herein.

a. Bacterial Infections

Although bacteria may not be harmful, and in some cases may bebeneficial, bacteria may also lead to infection. Bacterial infectionscan affect multiple organs and body systems including, but not limitedto, gastrointestinal tract, intestines, skin, mucous membranes, blood,lungs, kidneys, urinary tract, eyes, heart, meninges, respiratory tract,genitals, stomach, bone, connective tissue, and tissue surroundingorgans. Bacterial infections may affect more than one organ or bodysystem. Bacterial infections may be systemic. Bacterial infections maybe asymptomatic. Bacterial infections may cause a variety of symptomsincluding, but not limited to, fever, inflammation, wounds that do notheal, weeping wounds, skin rash, red bumps on the skin, abscesses,swollen lymph nodes, nausea, diarrhea, headaches, earaches, sore throat,fatigue, low blood pressure, hyperventilation, weak and rapid pulse,local or systemic pain, and muscle aches. Bacterial infections may causedeath. Subjects with co-morbidities or a compromised immune system maybe more susceptible to bacterial infections.

The diagnosis of a bacterial infection may include, but are not limitedto, symptomatic diagnostics, microbial culture, microscopy, biochemicaltests, PCR based diagnostics, and metagenomics sequencing. A microbialexamination may include sample collection, microbial cultivation,identification, and test of antibiotic susceptibility. The diagnosis mayinclude gram staining of the bacterial culture. The diagnosis mayinclude a coagulase test of the bacterial culture. The diagnosis mayinclude a catalase test of the bacterial culture. The diagnosis mayinclude blood tests. The blood tests may include, but are not limitedto, a full blood count, measurement of C-reactive protein, measurementof procalcitonin, and measurement of rapid plasma reagin. The diagnosismay include ELISA. The diagnosis may include PCR. The sample may begrown on an agar plate. The sample may be grown in nutrient broth. Thegrowth conditions may include varying factors (e.g., type of growthmedium, nutrients, selective compounds, antibiotics, temperature, pHlevel, oxygen level) to determine the type of bacteria growing. Thedetermination of bacteria growing on an agar plate or in a nutrientbroth may determine the bacteria responsible for the subject'sinfection. Discs containing antibiotic compounds may be placed on theagar plates. The antibiotic compounds may kill the bacteria growing onthe plate. The antibiotics that are effective at killing the bacteriamay aid in diagnosing the type of bacterial infection.

Samples for diagnosing a bacterial infection may be obtained from thesubject in need of treatment. The sample for testing may be from thesite of the infection. A sample for testing may be obtained from thesubject by swabbing of the skin, throat, or nose. A sample for testingmay be obtained from the subject by collecting pus or fluids fromwounds, abscesses, or other skin infections. A sample for testing may beobtained from the subject by collecting body fluids. The body fluids mayinclude blood, sputum, urine, and/or other body fluids. Multiple samplesmay be taken from the subject. Multiple samples may be taken around thesite of a prosthesis or medical device.

i. Clostridium difficile Infections

The bacterial infection may be Clostridium difficile. When stressed,Clostridium difficile may produce spores. The Clostridium difficilespores may be able to tolerate extreme conditions that the activebacteria cannot tolerate. A bacterial spore may make the bacteria moreresistant to environmental factors or induced factors that the bacteriamay be subjected to. Spores may help bacteria survive by being resistantto extreme changes in the bacteria's habitat. Extreme changes in thebacteria's habitat may include extreme temperatures, lack ofmoisture/drought, or being exposed to chemicals and radiation. Bacterialspores may be able to survive at low nutrient levels. Bacterial sporesmay be resistant to antibiotics and disinfectants. Bacterial spores maybe resistant to elimination. Bacteria that produce spores may bepathogenic. Spore-forming bacteria may be in the Bacillus andClostridium species. Spore-forming bacteria may be found in otherspecies of bacteria. There are different types of spores, including butnot limited to endospores, exospores, and spore-like structures calledmicrobial cysts. Spores may aid the bacteria in survival and serve asprotection for the cell.

Clostridium difficile may be transmitted from person to person by thefecal-oral route. Clostridium difficile may be shed in feces. Anysurface, device, or material (e.g., toilets, bathing tubs, andelectronic rectal thermometers) that becomes contaminated with feces mayserve as a reservoir for the Clostridium difficile spores. Clostridiumdifficile spores may be transferred to subjects via the hands ofhealthcare personnel who have touched a contaminated surface or item.Clostridium difficile may live for long periods of time on surfaces.Clostridium difficile spores may be heat-resistant. Clostridiumdifficile may not be not killed by alcohol-based hand cleansers orroutine surface cleaning. Clostridium difficile spores may survive inclinical environments for long periods. Once spores are ingested, theiracid-resistance may allow them to pass through the stomach unscathed.The Clostridium difficile spores may germinate and multiply intovegetative cells in the colon upon exposure to bile acids.

Symptoms of a Clostridium difficile infection may include, but are notlimited to watery diarrhea, fever, loss of appetite, nausea, abdominalpain/tenderness. Conditions that may result from a Clostridium difficileinfection may include, but are not limited to pseudomembranous colitis(PMC), toxic megacolon, perforations of the colon, and sepsis. AClostridium difficile infection may be deadly.

Antibiotic therapy for various infections may have the adverse effect ofdisrupting the normal balance of the gut flora. Clostridium difficilemay grow in the presence of an antibiotic. Clostridium difficile maygrow in the absence of other bacteria. The growth of Clostridiumdifficile may cause a Clostridium difficile infection in a subject.Administering a vaccination against Clostridium difficile to a subjectmay prevent or treat a Clostridium difficile infection.

The treatment or prevention of Clostridium difficile infections maycomprise immunization. After immunization, the subject may develop theability to quickly respond to a subsequent encounter with an immunogenbecause of immunological memory. This may be a function of the adaptiveimmune system. Therefore, by exposing a subject to an immunogen in acontrolled way, the subject's body may protect itself in the presence ofan immunogen. The immunogen may be a Clostridium difficile immunogen.The immunization may fortify a subject's immune system againstClostridium difficile infections. Immunizing a subject with Clostridiumdifficile spores may prepare the subject's immune system to respond toClostridium difficile. Immunizing a subject with Clostridium difficilespores may prevent a Clostridium difficile infection. Immunizing asubject with a Clostridium difficile spores may treat a Clostridiumdifficile infection.

The treatment or prevention of Clostridium difficile infections maycomprise immunization with spores formed by the NTCD_Tcd169 strain. Thetreatment or prevention of Clostridium difficile infections may compriseimmunization with spores formed by the NTCD_Tcd138 strain. The treatmentor prevention of Clostridium difficile infections may compriseimmunization with spores formed by the NTCD_Tcd169 strain in combinationwith spores formed by the NTCD_Tcd138 strain. Immunization with thespores disclosed herein may increases levels of anti-TcdA and anti-TcdBIgG antibodies in the subject.

Immunization may be through various techniques. The route ofimmunization may include, but is not limited to oral, nasal mucosal,sublingual, subcutaneous, intramuscular, intradermal, or Immunizationmay be through oral vaccination. Vaccines against bacteria that causeinfections may prepare the subject's immune system, thus helping tofight or prevent an infection. The vaccine may comprise the sporesdisclosed herein. The vaccine may comprise the spores disclosed hereinand a pharmaceutically acceptable excipient. The vaccine may comprisesynthetic oligodeoxynucleotides (ODNs). The synthetic oligonucleotidesmay comprise unmethylated CpG motifs (CpG ODNs) trigger cells thatexpress Toll-like receptor 9 to mount an innate immune response. CpGODNs may improve the function of professional antigen-presenting cells.CpG ODNs may boost the generation of humoral and cellularvaccine-specific immune responses.

In some embodiments, the vaccination is an oral vaccination. The oralvaccination may comprise Clostridium difficile spores. Oral vaccinationmay be the most effective method of protecting the gut againstinfection. Oral vaccination may expose the vaccination to proteolytic orhydrolyzing digestive enzymes, bile salts, extreme pH, rapid movement ofcontents, and limited access to the mucosal wall.

b. Modes of Administration

Methods of treatment may include any number of modes of administeringthe disclosed Clostridium difficile spores. Modes of administration mayinclude tablets, pills, dragees, hard and soft gel capsules, granules,pellets, aqueous, lipid, oily or other solutions, emulsions such asoil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups,elixirs, solid emulsions, solid dispersions or dispersible powders. Forthe preparation of pharmaceutical compositions for oral administration,the agent may be admixed with commonly known and used adjuvants andexcipients such as for example, gum arabic, talcum, starch, sugars (suchas, e.g., mannitose, methyl cellulose, lactose), gelatin, surface-activeagents, magnesium stearate, aqueous or non-aqueous solvents, paraffinderivatives, cross-linking agents, dispersants, emulsifiers, lubricants,conserving agents, flavoring agents (e.g., ethereal oils), solubilityenhancers (e.g., benzyl benzoate or benzyl alcohol) or bioavailabilityenhancers (e.g. Gelucire®). In the pharmaceutical composition, the agentmay also be dispersed in a microparticle, e.g. a nanoparticulatecomposition.

For parenteral administration, the agent can be dissolved or suspendedin a physiologically acceptable diluent, such as, e.g., water, buffer,oils with or without solubilizers, surface-active agents, dispersants oremulsifiers. As oils for example and without limitation, olive oil,peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil maybe used. More generally, for parenteral administration, the agent can bein the form of an aqueous, lipid, oily or other kind of solution orsuspension or even administered in the form of liposomes ornano-suspensions.

c. Combination Therapies

Additional therapeutic agent(s) may be administered simultaneously orsequentially with the disclosed Clostridium difficile spores. Sequentialadministration includes administration before or after the disclosedClostridium difficile spores. In some embodiments, the additionaltherapeutic agent or agents may be administered in the same compositionas the disclosed Clostridium difficile spores. In other embodiments,there may be an interval of time between administration of theadditional therapeutic agent and the disclosed Clostridium difficilespores. In some embodiments, administration of an additional therapeuticagent with a disclosed compositions and vaccines may allow lower dosesof the other therapeutic agents and administration at less frequentintervals. When used in combination with one or more other activeingredients, the Clostridium difficile spores of the present inventionand the other active ingredients may be used in lower doses than wheneach is used singly. Accordingly, the Clostridium difficile spores ofthe present invention include those that contain one or more otheractive ingredients, in addition to Clostridium difficile spores. Theabove combinations include combinations of Clostridium difficile sporesof the present invention not only with one other active compound, butalso with two or more other active compounds.

d. Evaluation of Treatment

The efficacy of the methods of treatment with Clostridium difficilespores disclosed herein may be measured. The status of the bacterialinfection may be monitored. The efficacy of the methods of treatmentdisclosed herein may be evaluated by the same or similar methods as usedfor diagnosis of the bacterial infection.

Evaluating the efficacy of the methods of treatment with the Clostridiumdifficile spores disclosed herein or monitoring the bacterial infectionmay include, but are not limited to, symptomatic diagnostics, microbialculture, microscopy, biochemical tests, PCR based tests, andmetagenomics sequencing. A microbial examination may include samplecollection, microbial cultivation, identification, and test ofantibiotic susceptibility. The evaluation or monitoring may include gramstaining of the bacterial culture. The evaluation or monitoring mayinclude a coagulase test of the bacterial culture. The evaluation ormonitoring may include a catalase test of the bacterial culture. Theevaluation or monitoring may include blood tests. The blood tests mayinclude, but are not limited to, a full blood count, measurement ofC-reactive protein, measurement of procalcitonin, and measurement ofrapid plasma reagin. The evaluation or monitoring may include ELISA. Theevaluation or monitoring may include PCR. The sample may be grown on anagar plate. The sample may be grown in nutrient broth. The growthconditions may include varying factors (e.g., type of growth medium,nutrients, selective compounds, antibiotics, temperature, pH level,oxygen level) to determine the type of bacteria growing. The presence,decreased presence, or lack of bacteria growing on an agar plate or in anutrient broth may determine that the bacterial infection is improvingor has been eradicated.

Samples for determining the efficacy of the methods of treatment withthe Clostridium difficile spores disclosed herein or monitoring thebacterial infection, may be obtained from the subject. The sample fortesting may be from the site of the infection, or the site where theinfection was previously present. A sample for testing may be obtainedfrom the subject by swabbing of the skin, throat, or nose. A sample fortesting may be obtained from the subject by collecting pus or fluidsfrom wounds, abscesses, or other skin infections. A sample for testingmay be obtained from the subject by collecting body fluids. The bodyfluids may include blood, sputum, urine, and other body fluids. Multiplesamples may be taken from the subject. Multiple samples may be takenaround the site of a prosthesis or medical device.

The evaluation of the efficacy of methods of treatment with theClostridium difficile spores disclosed herein or monitoring of thebacterial infection may indicate that the subject requires continuedtreatment with Clostridium difficile spores disclosed herein. Theevaluation of the efficacy of methods of treatment with Clostridiumdifficile spores disclosed herein or monitoring of the bacterialinfection may indicate the eradication of the bacterial infection in thesubject. The eradication of the bacterial infection may indicate thatthe subject no longer requires treatment with the Clostridium difficilespores disclosed herein.

6. KITS

The Clostridium difficile spores may be included in kits comprising thecompositions and vaccines; and information, instructions, or both thatuse of the kit will provide treatment for medical conditions in mammals(particularly humans). The kit may include an additional pharmaceuticalcomposition for use in combination therapy. The kit may include buffers,reagents, or other components to facilitate the mode of administration.The kit may include materials to facilitate oral administration. The kitmay include materials to facilitate nasal mucousal administration. Thekit may include materials that facilitate sublingual administration. Theinformation and instructions may be in the form of words, pictures, orboth, and the like. In addition or in the alternative, the kit mayinclude the medicament, a composition, or both; and information,instructions, or both, regarding methods of application of medicament,or of composition, preferably with the benefit of treating or preventingmedical conditions in mammals (e.g., humans).

The Clostridium difficile spores of the invention will be betterunderstood by reference to the following examples, which are intended asan illustration of and not a limitation upon the scope of the invention.

7. EXAMPLES Example 1. Construction of Tcd169

A recombinant fusion protein, designated mTcd138 (FIGS. 1B, 2) wasgenerated. mTcd138 contains the glucosyltransferase and cysteineproteinase domains of TcdB and the RBD of TcdA. Protein mTcd138 wasexpressed it in Bacillus megaterium. To ensure mTcd138 is atoxic, twopoint mutations (FIGS. 1B, 2) were made in the glucosyltransferasedomain of TcdB, which essentially eliminates mTcd138 toxicity in vitroand in vivo. To enhance the immunogenicity, mTcd138 was fused with theRBD of TcdB bridged with an amino acid linker (for example, GGSG),resulting in protein Tcd169 (FIGS. 1C, 3). The Chimeric DNA encodingTcd169 was ligated into B. megaterium expression vector which adds aC-terminal His-tag to the chimeric proteins. Tcd169 was purified frombacterial lysate by Ni-affinity chromatography and gel filtration (FIG.4).

Example 2. Tcd169 Immunization Induces Protective Responses Against BothToxins and Infection with an Epidemic C. difficile Strain

Immunization with Tcd169 via intraperitoneal (i.p), intramuscular (i.m.)routes induced similar levels of IgG antibody responses against bothtoxins (FIG. 5A). Tcd169 immunization induced potent neutralizingantibodies against both toxins (FIGS. 5B & 5C). Protection efficacy ofTcd169 immunization was evaluated in a mouse model of CDI. After threeimmunizations (10 μg Tcd169 per immunization with Alum as adjuvant, at14-day intervals) via i.p. or i.m. route, mice were challenged with 10⁶spores of C. difficile UK6 (BI/NAP1/027). In vehicle-immunized mice,significant disease symptoms including weight loss (FIG. 5E), severediarrhea in all mice; approximately 40% of mice succumbed by day 3 (FIG.5D). In contrast, all Tcd169-immunized mice survived (FIG. 5D) andshowed no signs of weight loss (FIG. 5E).

Example 3. Expression of mTcd138 in Non-Toxigenic C. difficile 85 Strain(NTCD), Resulting in Strain NTCD_Tcd138

By engineering NTCD to express the mTcd138 construct, two independentmethods of reducing CDI were combined in one treatment. The geneencoding mTcd138 was cloned in the E. coli-C. difficile shuttle vectorpRPF144 in E. coli Stb12 (Invitrogen). Conjugative transfer of plasmidfrom E. coli to NTCD was performed. Intermediate E. coli Stb12 harboringthe conjugative plasmid pRPF144-mTcd138 was used as a donor strain.Expression of mTcd138 in NTCD was verified by western blot analysis(FIG. 6).

Example 4. Oral Immunization of Mice with NTCD_Tcd138 Spores InducesMucosal and Systemic Toxin-Specific Antibody Responses, and ProtectsMice from Infection with a Hyper-Virulent C. difficile Strain

Oral immunization of mice with NTCD_Tcd138 (2×10⁶ spores perimmunization for 3 times at 14-day intervals) induced both IgG and IgAantibody responses specific for both toxins in sera (FIGS. 7A & 7B), aswell as IgA antibodies specific for both toxins in feces (FIG. 7C).NTCD_Tcd138 immunization also induced neutralizing antibodies againstboth toxins (FIG. 8). To determine whether NTCD or NTCD_Tcd138immunization can induce anti-C. difficile responses, a fusion proteincontaining full-length of C. difficile flagellin proteins FliC and FliDwere generated (designated FliCD), and measured anti-FliCD antibodylevels in sera and feces from NTCD- or NTCD_Tcd138-immunized mice. Itwas found that in comparison with NTCD, NTCD_Tcd138 immunization couldinduce higher levels of anti-FliCD IgG/IgA responses in both sera (FIGS.9A & 9B) and feces (FIGS. 9C & 9D). Protection efficacy of NTCD_Tcd138was further evaluated in a mouse model of CDI. After three oralimmunizations (2×10⁶ spores per immunization for 3 times at 14-dayintervals), mice were challenged with 10⁶ spores of C. difficile UK6. Invehicle (PBS)-immunized mice, significant disease symptoms includingweight loss (FIG. 10B) and severe diarrhea (FIG. 10C) were evident inall mice; approximately 20% of mice succumbed by day 4 (FIG. 10A). Incontrast, NTCD_Tcd138-immunized mice were fully protected and showed nosigns of disease at any stage (FIG. 10). Immunization with NTCD-onlyshowed slight, but not significant protection (FIGS. 10A & 10B) againstC. difficile challenge. NTCD_Tcd138-immunized mice secreted asignificantly less amount of toxins compared to NTCD-only or PBSimmunization groups (FIG. 11).

Example 5. Oral Immunization of Hamsters with NTCD_Tcd138 Spores InducesProtective Responses Against Both Toxins and Infection with aHyper-Virulent C. difficile Strain

The immunogenicity and protection efficacy of NTCD_Tcd138 was evaluatedin hamsters. Oral immunization of hamsters with NTCD_Tcd138 (2×10⁶spores per immunization for 3 times at 14-day intervals) induced similarlevels of anti-TcdA and anti-TcdB IgG antibodies in sera (FIG. 12).Anti-TcdA/TcdB IgA antibodies could not be measured due to the lack ofhamster-raised anti-IgA antibodies. Neutralizing antibodies against bothtoxins were detected in both sera and feces (FIG. 13). Hamsters areextremely sensitive to C. difficile infection, and usually die within 2to 3 days of infection at a dose of 100 spores. Therefore, hamster is anideal animal to test the strength of vaccine candidates against CDI. Toevaluate the protection strength of NTCD_Tcd138, the immunized hamsterswere challenged (2×10⁶ spores of NTCD_Tcd138 or NTCD per immunizationfor 3 times at 14-day intervals) with a hypervirulent C. difficilestrain UK6 at 2×10⁴ spores/hamster, which is 200-fold of the lethal C.difficile infection dose (100 spores). Oral immunization withNTCD_Tcd138 spores provided significant protection to hamsters againstsuch a high challenge dose (FIG. 14). In agreement with results in mice(FIGS. 11A & 11B), immunization of hamsters with NTCD-only spores alsoprovided protection though not significant (FIG. 15).

Example 6. Rapid Identification of Toxigenic/Non-Toxigenic C. difficileStrains by Multiplex PCR

To rapidly identify toxigenic/non-toxigenic C. difficile strains, asimple and fast 3-plex PCR method was developed to identify tcdA, tcdBand 16s rDNA specific for C. difficile. In this method, 5 μl of 12-24hrs of C. difficile culture was used as template (FIG. 15). This methodwill be used to distinguish toxigenic C. difficile strains fromnon-toxigenic C. difficile strains.

Example 7. Establishment of Novel and More Efficient Mouse Model of C.difficile Toxin Exposure

A mouse model of C. difficile toxin exposure was developed. A 5F infantfeeding tube catheter with side ports (Mallinckrodt Inc., St. Louis,Mo.; catalogue no. 85771) was inserted 2.5 cm up the colon. At thispoint, 100 μl of TcdA (10 μg)+TcdB (10 μg) or PBS was slowlyadministered over 30 s while pressure was applied to the anal area toprevent leakage. Following injection of the solution, the tube wasslowly removed and the rectal pressure was maintained for a further 30s. Four hours later, mice were euthanized and dissected to analyze thetoxin-mediated effects on the colon. The administration of TcdA/TcdBtriggered dramatic colonic inflammation (FIG. 16) and neutrophil andmacrophage infiltration. This “intra-rectal toxin instillation” approachwill be used to determine immunization protection against toxinchallenge via rectum.

Example 8. Construct Strain NTCD_Tcd169

The gene encoding Tcd169 was cloned in the E. coli-C. difficile shuttlevector pRPF144 in E. coli Stb12 (Invitrogen). Conjugative transfer ofplasmids from E. coli to non-toxigenic C. difficile 85 (NTCD) wasperformed. Intermediate E. coli Stb12 harboring the conjugative plasmidpRPF144-Tcd169 was used as a donor strain. Expression of Tcd169 in NTCDwill be verified by western-blot analysis. The resultant strains will bedesignated NTCD_Tcd169. The spores of these strains will be prepared.

Example 9. Determine Protection Against Systemic Toxin Challenge

A potent antibody response will be generated, that protects mice againstchallenge with a lethal dose of TcdA/TcdB (100 ng for each toxin). Oneweek after the third immunization, mice will be challenged IP with alethal dose of TcdA, TcdB or a mixture of TcdA and TcdB (100 ng for eachtoxin), and monitored for 72 hrs. If the protection is not optimal, doseoptimization experiments will be performed.

For oral immunization with NTCD_Tcd169 spores and adjuvants will bemixed and given to mice by gavage.

Example 10. In Vitro Neutralization Titers for Both Systemic and MucosalAntibodies, and Anti-Adherence Capability of Antibody Against Adhesionof C. difficile to Intestinal Epithelial Cells

The neutralizing titers against TcdA and TcdB, and anti-adherencecapability of sera and mucosal samples against adhesion of C. difficileto intestinal epithelial cells will be determined.

Example 11. Protection Against Systemic Challenge of the Toxins

Protection against systemic toxin challenge will be performed. LD₅₀imwill be used as the standard challenge dose to assess the levels of theprotection against systemic toxin challenge induced by the mucosalimmunization for each immunogen. The mucosal immunizations may induce asimilar level of protection as do parenteral immunization, in which 50%of mice will survive from challenge with LD₅₀im dose of each wild typetoxin, or two toxins given together. Should greater than 50% of micedie, a dose optimization will be performed as described below.

Example 12. Protection Against Mucosal Challenge with Toxins

In the above experiments, the generation of mucosal IgA and IgGantibodies against toxins will be examined. It will be assessed whetherthese antibodies produced in the gut can protect mice againsttoxin-mediated destruction of the mucosa. The “intra-rectal toxininstillation” approach will be used.

One week after the third immunization, mice (immunized with immunogensor placebo) will be directly injected with 10 μg of TcdA or TcdB or both(10 μg each) in a volume of 100 μl via rectum. Four hours later, micewill be euthanized and the colon will be carefully removed.Toxin-induced fluid accumulation will be quantitated as the ratio ofweight to length. In addition to assessing the fluid accumulation, thepathological signs, such as neutrophil infiltration and villus damage,will be evaluated histologically. Histopathological and neutrophilmyeloperoxidase (MPO) activity assays will be performed to evaluatemucosal damage and neutrophil infiltration. The resected colons will befixed in 4% formaldehyde buffered with PBS and then embedded withparaffin. Deparaffinized 6-μm-thick sections will be stained withhaematoxylin and eosin (H&E) for histological analysis, and the tissueinjuries will be blindly scored by a histologist. Histological gradingcriteria will be as follows: 0, minimal infiltration of lymphocytes,plasma cells, and eosinophils; 1+, mild infiltration of lymphocytes,plasma cells, neutrophils, and eosinophils plus mild congestion of themucosa with or without hyperplasia of gut-associated lymphoid tissue;2+, moderate infiltrations of mixed inflammatory cells, moderatecongestion and edema of the lamina propria, with or without goblet cellhyperplasia, individual surface cell necrosis or vacuolization, andcrypt dilatation; 3+, severe inflammation, congestion, edema, andhemorrhage in the mucosa, surface cell necrosis, or degeneration witherosions or ulcers. To measure MPO activities, a portion of the resectedcolon will be homogenized in 1 ml of 50 mM potassium phosphate bufferwith 0.5% hexadecyl trimethyl ammonium bromide and 5 mM EDTA. MPOactivities in a centrifuged supernatant will be determined using asubstrate o-phenylenediamine in a solution containing 0.05% of H₂O₂followed by measuring absorbance at 490 nm.

Example 13. Dose Optimization

Dose optimization of antigens will follow by performing doubling andhalving the optimized doses determined in previous examples for 4immunogens. If an adjuvant is used, e.g. dmLT, the same amount of theadjuvant will be mixed together with the immunogen before injection. Foreach dose and route of immunization, both systemic and mucosal IgG andIgA response will be monitored and their neutralizing titers will bemeasured. The lowest amount of antigen required to induce the highestlevel of serum and mucosal antibody response for each immunogen will beestablished.

Example 14. Protection Against Recurrent CDI in Mice

CDI has become increasingly difficult to manage due, in part, to theineffectiveness of current antibiotic regimens which are associated withhigh relapse rates. The efficacy of top-ranked regimens of immunizationin preventing disease recurrence in a spore-induced mouse CDI recurrencemodel which was developed previously will be evaluated. To induce CDIrelapse, surviving mice will be given antibiotic cocktail treatmentfollowed by oral gavage of C. difficile UK6 spores (10⁶/mouse) 30 daysafter the primary infection. The immunization and challenge scheme isillustrated in FIG. 17A. To assess whether immunization also protectsagainst disease relapse in naïve animals that recovered from CDI,surviving mice will be immunized after their recovery from the initialCDI as illustrated (FIG. 17B).

Example 15. Protection Against CDI in Hamsters

After three immunizations, hamsters will be pretreated with clindamycinfollowed by challenged with 100 to 10⁴ C. difficile UK6 spores. Weightchanges, diarrhea and modality will be recorded. After infection, fecalsamples will be collected for 10 days to compare spore secretion andtoxin levels in feces from immunized and non-immunized groups.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the present disclosureare set out in the following numbered clauses:

Clause 1. A non-toxigenic Clostridium difficile strain comprising:

a) an immunogenic protein comprising

-   -   i) a glucosyltranferase domain of Clostridium difficile toxin        TcdB;    -   ii) a cysteine proteinase domain of Clostridium difficile toxin        TcdB; and    -   iii) a receptor binding domain of Clostridium difficile toxin        TcdA,

wherein the glucosyltranferase domain of Clostridium difficile toxinTcdB comprises a W102A amino acid substitution and a D288N amino acidsubstitution.

Clause 2. The non-toxigenic Clostridium difficile strain of clause 1,wherein the immunogenic protein comprises the amino acid sequence of SEQID NO.: 4.

Clause 3. The non-toxigenic Clostridium difficile strain of clause 2,wherein the strain is non-toxigenic Clostridium difficile strain 138(NTCD_Tcd138).

Clause 4. The non-toxigenic Clostridium difficile strain of clause 1,wherein the Clostridium difficile form spores.

Clause 5. A non-toxigenic Clostridium difficile strain comprising:

i) a glucosyltranferase domain of Clostridium difficile toxin TcdB;

ii) a cysteine proteinase domain of Clostridium difficile toxin TcdB;

iii) a receptor binding domain of Clostridium difficile toxin TcdA; and

iv) a receptor binding domain of Clostridium difficile toxin TcdB,

wherein the glucosyltranferase domain of Clostridium difficile toxinTcdB comprises a W102A amino acid substitution and a D288N amino acidsubstitution.

Clause 6. The non-toxigenic Clostridium difficile strain of clause 5,wherein the immunogenic protein comprises the amino acid sequence of SEQID NO.: 3.

Clause 7. The non-toxigenic Clostridium difficile strain of clause 6,wherein the strain is non-toxigenic Clostridium difficile strain 169(NTCD_Tcd169).

Clause 8. The non-toxigenic Clostridium difficile strains of clause 5,wherein the Clostridium difficile form spores.

Clause 9. A vaccine comprising the Clostridium difficile spores ofclause 4 or clause 8 and a pharmaceutically acceptable excipient.

Clause 10. A vaccine comprising the Clostridium difficile spores ofclause 4 and clause 8 and a pharmaceutically acceptable carrier.

Clause 11. A method of treating or preventing Clostridium difficilebacterial infection in a subject in need thereof, the method comprisingadministering the vaccine of clause 9 or clause 10.

Clause 12. The method of clause 11, wherein the vaccine is administeredorally.

Clause 13. The method of clause 11, wherein the vaccine increases levelsof anti-TcdA and anti-TcdB IgG antibodies in the subject.

Clause 14. The method of clause 11, wherein the Clostridium difficilebacterial infection is caused by a hyper-virulent strain of Clostridiumdifficile.

Deposit Information.

Applicant deposited the non-toxigenic Clostridium difficile strainsNTCD_Tcd138 and NTCD_Tcd169, with the American Type Culture Collection(ATCC), 10801 University Boulevard Manassas, Va. 20110 USA, incompliance with the Budapest Treaty and in compliance with 37 C.F.R. §§1.801-1.809 on Nov. 21, 2019. The ATCC Accession No. for NTCD_Tcd138 isPTA-126151 and the ATCC Accession NO. for NTCD_Tcd169 is PTA-126152.This deposit shall be made available to persons determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37CFR § 1.14 and 35 USC § 122.

What we claim is:
 1. A method of treating or preventing Clostridiumdifficile bacterial infection in a subject in need thereof, comprisingadministering to the subject a vaccine that comprises a non-toxigenicClostridium difficile strain comprising: a) an immunogenic proteincomprising i) a glucosyltransferase domain of Clostridium difficiletoxin TcdB; ii) a cysteine proteinase domain of Clostridium difficiletoxin TcdB; iii) a receptor binding domain of Clostridium difficiletoxin TcdA; and iv) a receptor binding domain of Clostridium difficiletoxin TcdB, wherein the glucosyltransferase domain of Clostridiumdifficile toxin TcdB comprises a W102A amino acid substitution and aD288N amino acid substitution when compared to SEQ ID NO.: 5, andwherein the cysteine proteinase domain of Clostridium difficile toxinTcdB comprises a C698A amino acid substitution when compared to SEQ IDNO.:
 5. 2. The method of claim 1, wherein the glucosyltransferase domainof Clostridium difficile toxin TcdB is positioned immediately upstreamof the cysteine proteinase domain of Clostridium difficile toxin TcdB,wherein the amino acid sequence of the linked glucosyltransferase domainof Clostridium difficile toxin TcdB and the cysteine proteinase domainof Clostridium difficile toxin TcdB is set forth in SEQ ID NO.: 9;wherein the receptor binding domain of Clostridium difficile toxin TcdAcomprises the amino acid sequence of SEQ ID NO.: 6; and wherein thereceptor binding domain of Clostridium difficile toxin TcdB comprisesthe amino acid sequence of SED ID NO.:
 7. 3. The method of claim 1,wherein the immunogenic protein comprises the amino acid sequence of SEQID NO.:
 3. 4. The method of claim 1, wherein the non-toxigenicClostridium difficile strain is non-toxigenic Clostridium difficilestrain 169 (NTCD_Tcd169).
 5. The method of claim 1, wherein thenon-toxigenic Clostridium difficile strain forms spores.
 6. The methodof claim 1, wherein the vaccine further comprises a pharmaceuticallyacceptable excipient or carrier.
 7. The method of claim 1, wherein thevaccine is administered orally or intramuscularly.
 8. The method ofclaim 1, wherein the vaccine increases levels of anti-TcdA and anti-TcdBIgG antibodies in the subject.
 9. The method of claim 1, wherein theClostridium difficile bacterial infection is caused by a hyper-virulentstrain of Clostridium difficile.